1. Field of the Invention
This invention pertains to a method of separating free from bound fractions in a solid phase column immunoassay procedure and to a novel immunochemical composite for use therein.
2. Description of the Prior Art
Solid phase radioimmunoassay (RIA) has become popular because the system in which both the antigen-antibody reaction and the separation of free and bound antigen can be achieved in a single step results not only in a simple and rapid RIA, but also eliminates a number of handling and other errors which are inherent in other separation techniques. K. J. Catt, H. D. Niall, and G. W. Tregear, Biochem. J., 100:31c (1966), originally used as solid phase materials powdered polymers bearing reactive thiocyanate groups (--N.dbd.C.dbd.S) capable of forming covalent linkages with antibodies. Antibodies coupled to cyanogen-bromide-activated dextran and cellulose particles came into vogue as a result of the work of Wide, Porath, and Axen [L. Wide and J. Porath, Biochem. Biophys. Acta, 130:257 (1966), R. Axen, J. Porath and S. Ernback, Nature (Lond.), 214:1302 (1967), and L. Wide, Acta Endocrinol. (Copenhagen) Suppl., No. 142:207 (1969)]. Today antibodies have been covalently attached to a variety of solid matrices such as agarose, glass, polyacrylamide, and even iron oxide powder. S. J. Siegel, W. F. Line, N. S. T. Yang, A. Kwong, and C. Frank, J. Clin. Endocrinol. Metab., 37:526 (1973), H. H. Weetall, Chem. Abst., 77:18064y (1972), P. H. Moore and L. R. Axelrod, Steroids, 20:199 (1972), and L. S. Hersh and S. Yaverbaum, Clinica Chemica Acta, 63:69 (1975).
Although in principle first antibody linked to a solid phase represents a simple and versatile separation procedure, there are a number of disadvantages with this approach. The two questions one must answer in regard to solid phase systems using primary antibodies are, first, the possible loss of antibody activity (titer) with consequent waste of valuable antisera and, secondly, the possible reduction of sensitivity which may result with solid systems as compared with those which can be achieved using the same antisera in an aqueous solution. Recently, A. E. Bolton and W. H. Hunter, Biochemica et Biophysica Acta, 329:318 (1973), reported that recovery of antibody activity tended to be higher in solid preparations of antisera to hapten and small peptides than to similar solid preparations of antisera to large molecular weight protein hormones. Likewise, antisera to haptens covalently coupled to solid matrices show little or no loss in assay sensitivity when compared to the uncoupled antibody system whereas there was a dramatic loss of sensitivity when antibodies to large protein hormones were tested. These authors concluded that any loss of assay sensitivity resulting from the use of chemically attached antisera is probably caused by steric hindrance of the larger antigens.
Therefore, one of the major disadvantages against the use of solid preparations of primary antibody in RIA systems is that this method is not universal. (The approach cannot be implemented equally well for large and small molecules.) Other disadvantages which apply to all primary solid phase RIA procedures are: one must pipette an accurate amount of antibody from an insoluble suspension of gel, the assay mixture must be mechanically agitated to assure proper mixing, and centrifugation is usually required to separate the bound and free fractions.
The use of Sephadex columns in plastic syringes to bind a mixture of labeled I.sup.125 -thyroxine (T.sub.4) and T.sub.4 released from serum proteins by alkali, followed by a subsequent protein binding analysis with a fixed, limiting amount of thyroid binding globulin (TBG) on each column has recently been introduced. H. Seligson and D. Seligson, Clin. Chim. Acta, 38:199 (1972) and N. M. Alexander and J. F. Jennings, Clin. Chem., 20:553 (1974). In this procedure, the Sephadex column also serves to separate free T.sub.4 from the T.sub.4 -TBG complex. Similar tests have been developed for radiolabeled triiodothyronine (T.sub.3) in which an antibody to T.sub.3 is used as the competitive protein binder. N. M. Alexander and J. F. Jennings, Clin. Chem., 20:1353 (1974). In both assays the Sephadex column serves to bind the mixture of antigen and label while other serum constituents are not retained by the gel. Next, a fixed amount of competitive protein binder is incubated in the void volume of the column during which the antisera and label redistribute between the column and the binder. Elution with buffer removes the antigen binder complex while the free antigen remains attached to the Sephadex column. Although this approach is readily automated, this system requires the accurate loading of equal amounts of gel in all columns. Thus, one must continuously stir the Sephadex on a magnetic stirrer while transferring the suspension with a graduated pipette.
R. C. Boguslaski and C. L. Schwartz, Analytical Chemistry, Vol. 47, No. 9, 1583 (1975), have recently reported on a column radioimmunoassay for the determination of digitoxin (mw 765) which employs a column of immobilized primary antibody which acts both as a reaction chamber and separation device. Similar primary antibody column RIA's have been reported for vitamin B.sub.12 (mw 1,355), R. C. Boguslaski and C. P. Rathjen, Clinica Chemica Acta, 62:349 (1975), and insulin (mw .about. 6,000), J. W. Davis, J. M. Yoder, and E. C. Adams, Clinica Chemica Acta, 66:379 (1976). In view of the work of Bolton and Hunter, supra, this method is not universal in that assay sensitivity for large molecules (e.g., TSH having a molecular weight of 30,000) would be limited due to the properties of solid phase antibodies against large molecules.
It has been discovered that a universal solid phase column system for the separation of free and bound fractions can be obtained when one uses novel immunochemical composites containing a derivatized polysaccharide matrix and also containing positively charged imidoesters which covalently couple said derivatized polysaccharide matrix to antibodies.